CA1196911A - Process for the preparation of compounds containing carboxylic acid amide groups, in particular of peptides - Google Patents

Abstract

Abstract of the disclosure:
To prepare compounds containing carboxylic acid amide groups, in particular peptides, there are reacted compounds containing a carboxy group, in the presence of dialkylphosphinic acid anhydrides with compounds contaning a free amino group.

Description

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- 2 - HOE ~1/ 010 A great number of processes for preparing carboxy].ic acid amide and peptide bond~ has been proposed (cf., for example ~louben-Weyl, Methoden der organischen Chemie, vol.
XV, part II, pages 1 - 364; Angew. Chemie 92, 129 (1980).
~ll of these processes aim at ensuring the characteristi.cs which are required for the synthesis of peptides and have been more or less successful~ Specifically, they should be free from racemization, be realizable in simple and gentle manner while giving high yields and ~hile using easliy feasible, possibly harmless, starting products.

The present process provides.a new method of optimizing said conditions for an economic synthesis of peptides and amides.
It has now been found that compounds containing carb-oxylic acid amide groups, in parti.cular oligopeptides, can be prepared under gentle conditions in a high yield, by reacting compounds containing a free amino group, in particular aminocarboxylic acid derivatives or peptides, ~0 the carboxy groups of which are protected, in the presence of an anhydride of a dialkylphosphinic acid with colopounds containi.ng a f-ee carboxy group, in particular aminocarb-oxylic acids or peptides, the amino group of which is acylated The radicals introduced to protect the functional group may be split off subsequently in usual manner, in the case of the synthesis of peptides.
~y anhydrides of dialkylphosphinic acids there are to be underctood compounds of the formula I
P~ R

O = P - O - P = ~ I, 35 ~ R

~19~9~

- 3 - IIOE 81/F 010 wherein R is alkyl. The substituents ~ may be identical or different. Anhydrides wherein both P atoms carry the same substituents are preferred~
Particularly appropriates for the present invention S are anhydrides of the formula I wherein R i5 each time lower alky], preferably an alkyl having from 1 to 4 carbon atoms.
The dialkylphosphinic acid anhydrides used according to the present invention are colorless liquids. They are stable at room te~mperature and may be distilled under reduced pressure without being decomposed. They are readily soluble in most of the non-aqueous solvents, in particular lipid solvents such as chloroform or methy]ene chloride, but also in polar solvents such as dimethyl formamide and dimethyl acetamide.
Examples of suitable anhydrides of the dialkylphos-phinic acids are: methylethylphosphinic acid anhydride, methylpropylphosphinic acid anhydride, methylbutylphos-phinic acid anhydride, diethylphosphinic acid anhydride, di-n-propylphosphinic acid anhydride, di-n-butylphosphi--nic acid anhydride.
The dialkylphosphinic acid anhydrides may be prepared in known manner, for e~ample by reacting dialkylphos-phinic acid chlorides with dialkylphosphinic acid alkyl esters at 150 - 160C (cf. Houben-Weyl, Methoden der Orga-nischen Chemie, published by G. Thieme Verlag, Stuttgart 1963, volO XII, pages 22 and follow.). Particularly preferred are processes wherein dialkylphosphinic acid, salts or esters thereof are reacted with phosgen (cf.
German Patent No. 2,12~,583; German Offenlegungsschrift ~o. 2,225,545).
The reaction according to the present invention is carried out preferably in neutral or slightly alkaline medium. The ~ost simple way of proceeding consists in adjusting the pH of the medium a'~ the desired value by adding aliphatic or cycloaliphatic tertiary bases such

- 4 - HOE 81/F 010 as N-methylmorpholine, ~-ethylmorpholine or trialkylctmines having up to 6 carbon atoms in each alkyl group. T~hen operating with systems consistinc3 of waier and a solvent miscible with water there may be used, instead of the organic base, alkali metal salts acting as buffer systerns, for example salts of carbonic acid or of phosphoric acid.
Oligopeptides are prepared according to the process of the present invention by using as starting compounds on the one hand an amino acid or a peptide having a blocked carboxy group and on the other hand an amino acid or a peptide having a bloc};ed amino group.
~ ny protective groups that are commonly applied in the peptide synthesis may be used for the protection of the carboxy groups. Particularly appropriate are esters of straight-chain or branched aliphatic alcohols such as methanol, ethanol, tertiary butanol. Esters of arali-phatic alcohols such as benzyl alcohol or diphenylmethyl-carbinol may be used alternatively.
Any protective groups that are usual in the peptide synthesis may be used likewise for the protection of the amino groups. Particularly appropriate are, for example, the carbobenzoxy radical and the carbo-tertiary butyl-oxy radical.
Suitable solvents are all anhydrous inert solvents that are commonly applied in the peptide synthesis, for example methylene chloride, chloroform, dimethyl formamide, dimethyl acetamide, dioxan or tetrahydrofuran.
The synthesis may be carried out alternatively using systems consisting of water and an organic solvent miscible with water such as dioxan/water, tetrahydrofuran/water or dimethyl formamide/water. ~sing such systems is parti-cularly advantageous when connecting peptides that are predomillantl~r water-soluble.
The react;on proceeds gellerally in sufficiently ~1ick manner at room teinperatureO Slight heating is possible.

~196g~

- 5 - XOE 81/F 010 Higher temperatures, however, for example of more than 50C, are not recommended because of the danger OL race-mization, in particular in the peptide synthesis.
The dialkylphosphinic acid anhydrides according to the present invention are used preferably in e~cess(about 2 to 2.5 moles of dial~ylphosphinic acid anhydride per mol of pepti~e bond to be boun~. They may be added drop-wise to the reastion mixture as auch in undiluted state.
The process accordlng to the present invention is distinguished by a number of advantages, as compared to the actually common processes.
When using the dialkylphosphinic acid anhydrides there are left no difficultly soluble by-products upon completion - of the synthesis, and the process of the invention is there-fore superior over the peptide linkage forminy processes using dicyclohexylcarbodiir,~ide, which is fre-quently applied actually.
The dialkylphosphinic acid anhydrides are easier ob-tainable and easier to handle than most of the reagents commonly used for this purpose as far as they show little racemization.
As compared to the hitherto used processes for the peptide synthesis using activation agents based on tri-valent or pentavalent phosphorus, for example the peptide synthesis by the phosphorus azo method (cf. Liebigs Ann.
Chem. 580, page 68), synthesis methods using diethyl-chlorophosphite ox tetraethylpyrophosphite (cf. ~.Am. Chem.
Soc. 74, 5304 (1952) and J. Am. Chem. Soc. 74, 5307 and 5309 (1952) and the synthesis method using polyphosphoric acid esters (cf. Ber. 91, (1958), p. 1073 - 1082), the process according to the present invention has the advan-tage of showing little racemization.
The proces~ of the present invention can be carried out in simple manner and yields peptides in a high optical purity and in a high yields. It is moreover economical and litt]e polluting.

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-- 6 ~ HOE 31/F 010 The dialkylphosphinic acid anhydrides are of low molecular weight, they are easy to obtain and easy to purify and have a high portion of reactive yroups, per weight unity, as well as a good lipophility. The dialkyl-phosphinic acid anhydrides and the correspondiny dialkyl-phosphinic acids ar~ lipid-soluble~ This permits a workin~
up of water-soluble peptide derivatives via a first precipitation step using suitable lipoid solvents.
The dialkylphosphinic acid obtained from the dialkyl-phosphinic acid anhydride in the course of the peptidesynthesis may be recovered from the remaining solutions of the synthesis reactionO
The dialkylphosphinic acids may be recovered from a relatively great quantity of aqueous synthesis mother liquors by extracting with solvents such as chloroform and iso-butanol, followed by distillative working up, it being of particular technical interest that the dialkylphosphinic acids can be distilled in vacuo without being decomposed The recovered dialkylphosphinic acids may be converted easily into the corresponding dialkylphosphinic acid anhydrides by the process of German Offenlegungsschrift No. 2,2~5,5~5.
When operating with the system consisting of water and an organic solvent miscible with water as described hereinbefore, the organic base may be replaced by salts o~ carbonic acid or phosphoric acid showing an ~lkaline reaction in aqueous solution, which facilitates the above-described working up of the dialkylphosphinic acids follo-wing the synthesis. In this case the extraction step can be dispensed with. The dialkylphosphinic acid, upon liberatlon, can be directl~ separated by distillation from the evaporated mother liquor of the synthesis.
The present invention will be illustrated in the following examples.

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E X A M P L E
Z--Gly-Gly oEt A~ 0C, 7.0 g (0.0S mol) of H-Gly-OC2H5 HCl, ~5 ml of N-ethyl morpholine and 20 g of methylethylphosphinic acid anhydride were added successively to a solution of 10.5 g (0~05 mol) of carbobenzoxy-glycine ln 20 ml DMF, while stixring and cooling ~roughly. me mixture was b~ought tD
room temperature, whlle stirring. After 16 hours o~
standin~ at room temperature the ~olvent was distilled off in vacuo, and the residue was dissolved in a mixture of 200 ml of ethyl acetate and 100 ml of a 5 % potassium bisulfate solution. The ethyl acetate solution was washed twice with 100 ml each of saturared sodium bicarbonate solution, dried over sodium sulfate and evaporated in vacuo.
Yield: 12.0 g oi Z-dipeptide ester having a melting point of 80 ~80 ~ of the th.).

~ X A M P L E 2 Z-Val-TyrtButtHis-OCH3 At 0C, 30 mol of N-ethyl morpholine, 21~5 g of Z-Val-Tyr-tBut)-OH and 20 g of methylethylenephosphinic acid anhydride were added suscessively, while stirring, to a suspen~ion of 11.5 ~ of H-His-OCH3 HCl in 100 ml of dimethyl formamide. The reaction solution which was practically clear after the exothermic reaction had been completed was allowed to stand overnight at room tempera~
ture, thereater the solvents were evaporated in vacuo at room temperature, and a mixture of 100 ml of saturared NaHCO3 solution and 200 ml of acetic acid ethyl es~er was added to the residue. The crude product was introduced into the ethyl acetate phase, whil~ shakingl which phase was washed with a small amount of water, dried over 3S sodium ~ulate and then brou~ht to dryness in vacuo. The Z-tri~pep~ide ester remaining in the residue became solid upon digesting with diethyl ether.
.~ .
~ .

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Yield: 21 g, melting point 188 to 190 ~ ~D ~ 8.2 (c = 1, DMF). From the mother liquor there rnay be ob-tained another 2.5 g of the peptide by evapOratinCJ the sol-vent and recry~tallizing the mixture from acetic acid ethyl ester/diethyl ether. Total yie]d: 76 % of the theoryO

.. . . . ~_ .
_-Pro-Ala-Lys-(Boc)-Phe-NH~
19.1 Grams of H-Lys(Boc)-Phe-NH2 HCl (0.0~4 mol) were dissolved in 100 ml of dimethyl formamide, and at 0C 26 ml of N-ethyl morpholine, 16.0 g (0.005 mol) of Z-Pro-Ala-OH
and 17.5 g of methylethylphosphinic acid anhydride were added, while stirring. The reaction mixture was allowed to s-tand at room tempera-ture for 48 hours, thereafter it was brought to dryness in vacuo, the resiclue was digested wi'ch 100 ml of 2N sodium carbonate solution, 100 ml of a 10 ~
aqueous citric acid solution and 100 ml of dist. water and was then dried in vacuo over phosphorus pentoxide.
Yield: 30.4 g = 89 % of the th. ~]D = 27.0 (c=1 DMF); melt. point 163C.

E X A M P I. E 4 -Z-Asp(OBu )-Phe-NH
At 0C, 1.6 y (0.01 mol) of H-Phe-NH2 were dissolved, together with 28 ml of N-ethyl morpholine in 20 ml of dimethyl formamide. While stirring and cooling, 3.23 g ~ (0.01 mol) of Z-Asp(OBut)OH and~of methylethylphosphinic acid anhydride were added to said solution~ The reactlon solution thus prepared was allowed to s-tand overnight at room temperature.The product was worked up by extraction with ethyl acetate, washlng with water, aqueous sodium bicarbonate solution and with a 5 % aqueous KHSO~ solution, b~ concentrating the ethyl aceta-te solution dried over sodiuln - 9 ~ HOE 81/E 010 sulfate and by precipitating -the final product with diethyl ether.
Yield: 4.0 g (85 % of the th.), m.p. 162 ~ D =
-33.1 (c = 1, Cl130H) _ !, Z-Gly-Thr(But)Phe-OCH l ~ ,!
18.6 Grams (0.05 mol) of H-Thr(Bu )-Phe-OCH3 HCl, 30 ml (0.238 mol) of N-ethyl morpholine and 10.4 g (0.05 mol) of Z-Gly-OH were dissolved successively in 120 ml of dimethyl sulfoxide (p.a. Merck), and 4 g of methylethyl-phosphinic acid anhydride were added portionwise, whi]e stirring and cooling with ice and with the exclusion of moisture. Stirring was continued for another 24 hours at room temperature, and the reaction solution was then introduced into 500 ml of saturated sodiu~ bicarbona-te solution, whereupon the reaction product precipi';ated.
The supernatant solution was-decanted, and the precipitate was dissolved in acetic acid ethyl ester. The ethyl ace-tate solution was washed with water, dried over magne-sium sulfate, largely concentrated in vacuo, and the final product was precipitated with petroleum e-~her. It crystalliued overni~ht at +4C.
Yield: 1~.0 g (75 % of the theory); melting point:
95C; r~ ~D = +26 (c = 1, DMF).

Z Phe-cyclohexylamide __ _ 30 30 Gxams (0.01 mol) of Z-Phe-OH, 1.0 g (0O01 mol; 1.2 ml) of cyclohexylamine and 5 ml of N ethyl morpholine were dissolved in 30 ml bf DMF, and while cooling with ice and stirring, ~ g of methylethylphosphinic acid anhydride were added~ When the exothermic reaction had been completed (wi-th a-rise in temperature of up to ~10), the solution was a~lowed to reach room temperature, while stirring, and - 10 -- HOE 3~ 010 the reaction mixture was then worked up as has been des-cribed in E,cample 1.
Yield~ 3.3 g (85 % of the th.) ~JD = -2 . 8 (c = 1, DME) m.p. 167C

E X A_M P L E 7 ~ ~ ~e ~ U~
2.26 g (0.005 mol) of Z-Phe-Arg-OH and 1.55 g ~0.005 mol) of H-Trp-Gly-OCH3 HCl were dissolved in 10 ml of ~methyl formamide at 0C while adding 5 ml of N-e-thyl morpholine.
Next, 2 g of methylethylphosphinic acid anhydride were added dropwise while stirring at this temperature. Stirring was continued at room temperature for one hour, whereupon the batch was left to stand at room temperature for 20 hours. After separation of dimethyl formamide by distillatior.
in vacuo at room temperature, the residue was digestecl with 30 ml oE saturated sodium carbonate solution and the solids obtained were dried in vacuo over P2O5.
Yield: 3.0 g (90 % of the theory~.

H-Phe-Arg-Trp-Gly-OCH3 2 HCl 3 Grams of the carbobenzoxytetrapeptide were dissolved in 200 ml of methanol, the air in the reaction vessel was expelled by nitrogen and 1 g of a 10 % Pd/barium sulfate catalyst was added. Hydrogenation was carried out in usual manner by stirring and passing through hydrogen. The pH
~le~r~me ~ i~ IC
of the reaction solution, determined by ~ ~o~r~rie measurement, was maintained at 4.0 by adding a 1 N
methanolic hydrochloric acid solution~ The splitting off of the carbobenzoxy radical by hydrogenation was terminated after 2 and a half hours, which could be observed by the consulnption oE the 1 N methanolic HCl solut.ion and by a - ~ ~L9~,g~

~ EI~E 81/F 010 stop of the CO2 evalution. Next, hydroc~en was expelled from the reaction vessel by nitrogen, the solvents were suction-filtered from the catalyst under nitrogen, the solution was concentrated to dryness in vacuo at rcom temperature and the residue was digested wi-th absolute diethyl ether.
Yield: Upon drying in a high vacuum: 2.5 g (prac-tically quantitative).
r~ ~D = ~ 5-5 (c = 1, glacial acetic acid).

X A M P L E 8 a Determination of the degree of racemization by high pressure liquid chromatoc3raphy:
25 ~l of a 0.4 % solution of the peptide derivative of Example 8 were introduced into the separating column of a liquid chromatograph (dimensions of the column: 0.~ x 25 cm, packing Spherosil( ) XOA 600 Normatom( ) 5 ~Im) which had been equilibrated with a solvent mixture con-sisting of chloroform/methanol/water/forrnic acid/amrnonium 20 formate (900:400:30:7:2.5) and the chromatogram was devel-oped by pumpincJ the above-described solvent through the column at a rate of 2 ml per minute under a pressure of ~41 bar. The diastereomeric peptides separated on the column were determined quantitatively in the :Elow photometer, based on their absorption at 280 nm. The D-Ar~ diastereomer of the above prepared hydrochloride of the tetrapeptide ester was eluted under said conditions about 10 minutes after the column separation had started, the L-diastereomer appeared about 15 minutes :Later. The quan-tity of the D-Arc~-diastereomer amounted to 2 % of the hydrochloride of the tetrapeptide ester that had been introduced.

E X A M P L F. _9 Z--Trp-Gly-OC~13 At 0"C there were added while stirrinc~ 6.5 ml o~ N~methyl 3~ ~

- 12 - OE 81_F 010 morpholine to a mixture of 3.35 g ~0.05 mol) of Z-Trp-OH
and 1.25 g of H-Gly-OMe. Into the solution obtained there were added dropwise at the -temperature specif:ierl, while stirring, 4 g of metl1ylethylphosphinic acid anhydride.
The mixture was allowed to stand at room temperature for 10 hours and the solvent was separated by distillation in vacuo at room temperature. The residue was taken up by a mixture of 25 ml of water and 70 ml of ethyl acetate, the ethyl acetate extract was separated and the aqueous solution was extrac-ted twice using each time 10 ml of ethyl acetate. The combined ethyl acetate ext:racts were washed twice using each time 7 ml of water, a 5 % potassium sulfate solution (until an acid reaction took place) and a saturated NaHCO3 solution until a slightly alkaline reaction took place. Upon drying over magnesium sulfate and clearing with active carbon, the solvent was largely separated by distillation in vacuo and the residue was - digested with dilsopropyl etherO
Yield: Upon drying in vacuo over phospnorus pent--oxide: 2.8G g = 70 ~ of the theory;
colorless crystals.
Melting point: 157~C ~9~ 1D = -12.5 (c = 1, glacial acetic acid~.

-Z-Tr~G ~
To the suspension of 3.35 g (0.01 mol) of Z-Trp-OH and 1.25 g (0.01 mol) of H~Gly-OCH3 in 15 ml of dimethyl form~
amide there were added portionwise altogether 7 ml of water and subsequently 5.1 g of a -finely powdered sodium bicarbonate. The suspension was largely clarified by adding dropwise 4 ml of methyle-thy] phosphinic acid anhydride. A sample of the reaction solution diluted with water showed a neutral reaction. Upon heatin~ to room ter.1perature the solution was stirred overnight and the solvents were separated by distillation in a high - 13 - HOE_81/F 010 vaccum at room temperature. The residue was taken up in a mixture of 25 ml of water and 70 ml. of ethyl acetate, the e-thyl acetate extract was separa-ted and the aqueous phase was ex-tracted twice using each time 10 ml of ethyl acetate.
The combined ethyl acetate extracts were washed twice us.i.ng each time 7 ml of water, a 5 ~ potassium bisulfate solution (until an acid reacti.on took place) and a saturated sodium bicarbonate solution (until s slightly alkaline reaction took place). Upon drying over macJneSium sulfate the ethyl acetate solution was largely separated by distillation in vacuo and the residue was digested with diisopropyl ether.
Yield upon drying in vacuo over P2O5: 3.0 g (73 % of the theory).
15 Melting point: 157C ~D = -12.9 (c = 1, glacial acetic acid).

E ~ A M P L E 11 Z-Trp-Gly-OCH3 To a mixture of 3.35 g ~0.01 mol) of Z-Trp-OH, 1.25 g (0.01 mol) of H~Gly-OCH3 HCl and 15 ml of dimethyl form-amide there was added while stirring at 0C a soluti.on of 7.0 g of K3PO~ H2O in 8 ml of water. While further stirring there were added dropwise 4.0 ml of methyl-ethylphosphinic acid anhydride and the batch was subse-quently allowed to heat -to room temperature while stirring.
After a 24 hours' stirring at room ternperature the solvents were separated by di.stillation in vacuo at room temperature the residue was taken up in a mixture of 25 ml of water and 80 ml c,f ethyl acetate, the wa-ter phase left was extracted a second time with 20 ml of ethyl acetate and -the combined e-thy1 acetate extracts were shaken three -times UsincJ each time 8 ml of a 5 % potassium bisula-te solu-tion and a sa-turared sodium bicarbona-tc solu-ti.on. Upon drying the el-hyl ace-tate solution with sodium su]Latc, the product ~:~9~

was isolated by evaporating the solvents and di.gesting the .residue with a low-boiling petroleum ether (boiling point 40 - 80C).
Yield upon drying in a high vaccum over P2O5: 3.0 g (73 % of the theory), melting point: 157C
~ D = -12,9 (c = 2, gl~cial aceti.c acid).

Claims (6)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a compound containing a carboxylic acid amide group in which a compound containing a carboxy group is reacted, in the presence of a dialkylphosphinic acid anhydride, with a compound containing a free amino group and radicals that may have been introduced to protect other functional groups are then split off.

2. A process as claimed in claim 1 in which the compound containing a carboxylic acid amide group is a peptide.

3. A process as claimed in claim 1 in which the reaction is carried out in a medium consisting of water and an organic solvent miscible with water.

4. A process as claimed in claim 1, claim 2 or claim 3 in which the reaction medium is buffered with at least one member of the group of alkali metal salts of carbonic acid and phosphoric acid.

5. A process as claimed in claim 1, claim 2 or claim 3 in which the dialkylphosphinic acid anhydride has the formula wherein R is an alkyl group.

6. A process as claimed in claim 1, claim 2 or claim 3 in which the dialkylphosphinic acid anhydride is methylethylphosphinic acid anhydride.